Accumulation of polyethylene microplastics in river biofilms and effect on the uptake, biotransformation and toxicity of the antimicrobial triclosan.

The interaction of multiple stressors in freshwater ecosystems may lead to adverse effects on aquatic communities and their ecological functions. Microplastics (MPs) are a class of contaminants of emerging concern that can exert both direct and indirect ecotoxicological effects. A growing number of studies have investigated MPs-attached microbial communities, but the interaction between MPs and substrate-associated biofilm (i.e., on natural river substrates, such as stones and sediments) remains poorly studied. In this work, the combined effects of polyethylene MPs (PE-MPs) with a particle size of 10-45 µm (2 mg/L) and the antimicrobial triclosan (TCS) (20 µg/L) were investigated on river biofilms through a short-term exposure experiment (72 h). To the best of authors' knowledge, this is the first time that the combined effects of MPs and chemical contaminants in substrate-associated river biofilms were assessed. Different response parameters were evaluated, including (i) exposure assessment and ii) contaminants effects at different levels: bacterial community composition, antibiotic resistance, extracellular polymeric substances (EPS), photosynthetic efficiency (Yeff), and leucine aminopeptidase activity (LAPA). Triclosan was accumulated in river biofilms (1189-1513 ng/g dw) alongside its biotransformation product methyl-triclosan (20-29 ng/g dw). Also, PE-MPs were detected on biofilms (168-292 MP/cm2), but they had no significant influence on the bioaccumulation and biotransformation of TCS. A moderate shift in bacterial community composition was driven by TCS, regardless of PE-MPs co-exposure (e.g., increased relative abundance of Sphingomonadaceae family). Additionally, Yeff and EPS content were significantly disrupted in TCS-exposed biofilms. Therefore, the most remarkable effects on river biofilms were related to the antimicrobial TCS, whereas single PE-MPs exposure did not alter any of the evaluated parameters. These results demonstrate that biofilms might act as environmental sink of MPs. Although no interaction between PE-MPs and TCS was observed, the possible indirect impact of other MPs-adsorbed contaminants on biofilms should be further assessed.

Anaerobic treatment of swine manure under mesophilic and thermophilic temperatures: Fate of veterinary drugs and resistance genes.

The effect of anaerobic treatment of swine manure at 35 °C (mesophilic) and 55 °C (thermophilic) on methane production, microbial community and contaminants of emerging concern was investigated. Pasteurization pretreatment and post treatment was also investigated in combination with anaerobic treatment at 35 °C. Specific methane production (SMP), 26 pharmaceutical compounds (PhACs) and five antibiotic resistance genes (ARGs) (qnrS, tetW, ermB, sul1 and blaTEM) were evaluated. Mesophilic treatment resulted in the highest SMP regardless of whether pasteurization was applied. Marbofloxacin was the most abundant antibiotic in swine manure. In general, all groups of PhACs showed higher removals under thermophilic temperatures as compared to mesophilic. In general, pasteurization pretreatment followed by mesophilic anaerobic digestion provided the highest removals of ARGs. Finally, the genera Streptococcus, Clostridium and Pseudomonas which contain pathogenic species, were present in the swine manure. Streptococcus, which was the most abundant, was decreased during all the treatments, while the others only decreased under certain treatments.

Antimicrobial effect of Moringa oleifera seed powder against Vibrio cholerae isolated from the rearing water of shrimp (Penaeus vannamei) postlarvae.

Shrimp farming has experienced rising costs as a result of disease outbreaks associated with Vibrio spp. Suitable strategies for disease prevention and control are therefore urgently needed. This study aimed to evaluate the antimicrobial effect of Moringa oleifera seed powder against Vibrio cholerae in the rearing water of Pacific white shrimp (Penaeus vannamei) postlarvae. In vitro assays included the determination of minimum inhibitory concentration (MIC) of M. oleifera seed powder against V. cholerae, whereas in vivo assays included the effect of M. oleifera seed powder on bacterial load and water quality parameters in the rearing tanks, as well as its effect on shrimp postlarvae survival. M. oleifera seed powder inhibited the growth of V. cholerae with MIC values of 62·5 µg ml-1 . Moreover, seawater pH of treated tanks (8·66) was significantly lower (P < 0·01) than pH of the control tanks (9·02), whereas the visibility of treated tanks (37·08 cm) was significantly higher (P < 0·01) as compared to control tanks (35·37 cm). Likewise, V. cholerae load was significantly reduced (P < 0·01) from 4·7 × 104 to 3·1 × 103  CFU per ml in tanks treated with M. oleifera seed powder. Altogether, this study demonstrates the antimicrobial activity of M. oleifera against V. cholerae in shrimp culture.

Assessing the occurrence of pharmaceuticals and antibiotic resistance genes during the anaerobic treatment of slaughterhouse wastewater at different temperatures.

This study investigates the effect of psychrophilic, mesophilic and thermophilic temperatures on the anaerobic treatment of slaughterhouse wastewater, in terms of biogas production, occurrence of 30 pharmaceutical compounds of veterinary use, 4 antibiotic resistance genes (ARGs) which provide resistance to tetracyclines (tetW), fluoroquinolones (qnrS), macrolide-lincosamide-streptogramin (ermB) and sulfonamides (sul1) antibiotics, as well as class I integron-integrase gene (intI1), related to horizontal gene transfer. The highest methane yield was obtained at a mesophilic temperature (35 °C) (323 mL CH4/g TCOD) followed by the yield obtained at thermophilic temperature (53 °C) (242 mL CH4/g TCOD). Regarding pharmaceuticals, chlortetracycline, oxytetracycline, tilmicosin, and lincomycin were the most abundant in the slaughterhouse wastewater, being detected predominantly in the solid phase (with median concentrations >200 µg/kg dry weight). On the other hand, ciprofloxacin, ofloxacin, norfloxacin, lincomycin and ibuprofen were the most predominant in the anaerobic digestate regardless of the treatment temperature. Psychrophilic temperatures (21 °C) exhibited moderate to low pharmaceuticals removal, while a large fraction of them were removed at a thermophilic temperature reaching 70-90% removals for tetracycline, macrolides and one sulfonamide (sulfapyridine). The highest relative abundance of the quantified ARGs was found at 53 °C, suggesting that thermophilic temperatures normally associated with better removals of pathogens do not necessarily show better removals of antibiotic resistance genes.

High-throughput sequencing-based analysis of bacterial communities associated with Barbour's seahorses (Hippocampus barbouri) from Surigao del Norte, Philippines.

This study identified the bacterial community composition associated with Barbour's seahorses (Hippocampus barbouri). Seahorses and samples from the surrounding environment (sediment and water) were collected from Cantiasay Island in Surigao del Norte, Philippines. Genomic DNA was isolated from these samples, and the V1-V3 region of the 16S rRNA gene was amplified and sequenced on the Illumina MiSeq platform. There were 929 803 sequence reads corresponding to 859 operational taxonomic units (OTUs), which were obtained from the six genomic libraries. The results showed that the most abundant OTUs were affiliated to the phylum Proteobacteria, particularly those belonging to the genera Shewanella, Pseudomonas, Acinetobacter and Aeromonas, which were found to be dominant in seahorse gut samples. The only exception was for the skin of male seahorses, which was dominated by members belonging to the genus Bacillus (phylum Firmicutes). These findings on bacterial community structure and composition may shed light on therapeutic claims of Barbour's seahorses and help in the conservation of this species.

Use of bacteriophage vB_Pd_PDCC-1 as biological control agent of Photobacterium damselae subsp. damselae during hatching of longfin yellowtail (Seriola rivoliana) eggs.

AIMS: This study describes the effect of phage therapy on hatching of longfin yellowtail (Seriola rivoliana) eggs challenged with Photobacterium damselae subsp. damselae. METHODS AND RESULTS: A lytic phage (vB_Pd_PDCC-1) against P. damselae subsp. damselae was isolated and characterized. The use of phage vB_Pd_PDCC-1 increased the hatching rate of eggs, and reduced presumptive Vibrio species to non-detectable numbers, even in non-disinfected eggs. High-throughput 16S rRNA gene sequencing analysis revealed that phage vB_Pd_PDCC-1 caused significant changes in the composition and structure of the associated microbiota, allowing that members (e.g. those belonging to the family Vibrionaceae) of the class Gammaproteobacteria to be displaced by members of the class Alphaproteobacteria. CONCLUSIONS: To the best of our knowledge, this represents the first study evaluating phage therapy to control potential negative effects of P. damselae subsp. damselae during hatching of longfin yellowtail eggs. SIGNIFICANCE AND IMPACT OF THE STUDY: The Seriola genus includes several important commercial fish species due to its rapid growth and easy adaptability to confinement conditions. However, bacterial infections (especially those caused by Vibrio and Photobacterium species) are among the main limiting factors for the intensification of marine fish aquaculture, particularly during early development stages. Therefore, the use of phages, which are natural killers of bacteria, represents a promising strategy to reduce the mortality of farmed organisms caused by pathogenic bacteria.

In vitro assessment of potential probiotic characteristics of indigenous Lactococcus lactis and Weissella oryzae isolates from rainbow trout (Oncorhynchus mykiss Walbaum).

AIM: The objective of this study was to evaluate the probiotic potential of lactic acid bacteria (LAB) isolated from the intestinal ecosystem of rainbow trout. METHODS AND RESULTS: Among LAB isolates, 10 of them were selected and screened for resistance to acid and bile salts, pancreatin, sodium chloride and temperature, hydrophobicity, growth profile and antimicrobial activity against fish pathogens. Then, biosafety assessments were investigated. Selected LAB tolerated to gastrointestinal physiological conditions, pancreatin and a range of sodium chloride and temperature. They also exhibited hydrophobicity and showed antagonistic activity against Streptococcus iniae and Yersinia ruckeri. Results of 16S rRNA gene sequencing showed that selected LAB belonged to the Lactococcus lactis (n = 5) and Weissella oryzae (n = 5) species. They exhibited no ß-haemolytic activity, while six selected LAB were resistant to some antibiotics. None of them harboured virulence factors. CONCLUSIONS: This study revealed probiotic characteristics of indigenous LAB isolated from the intestinal ecosystem of rainbow trout. However, further studies are required to confirm the effectiveness of these isolates as probiotics in aquaculture. SIGNIFICANCE AND IMPACT OF THE STUDY: To the best of our knowledge, for the first time, the presence of probiotic candidates belonging to W. Oryzae was confirmed in fish intestinal microbiota.

Human exposure assessment to antibiotic-resistant Escherichia coli through drinking water.

Antibiotic-resistant bacteria (ARB) are a potential threat to human health through drinking water with strong evidence of ARB presence in post treated tap water around the world. This study examines potential human exposure to antibiotic-resistant (AR) Escherichia coli (E. coli) through drinking water, the effect of different drinking water treatments on AR E. coli and the concentration of AR E. coli required in the source water for the EU Drinking Water Directive (DWD) (Council Directive 98/83/EC, 0CFU/100ml of E. coli in drinking water) to be exceeded. A number of scenarios were evaluated to examine different water treatment combinations and to reflect site specific conditions at a study site in Europe. A literature search was carried out to collate data on the effect of environmental conditions on AR E. coli, the effect of different water treatments on AR E. coli and typical human consumption levels of tap water. A human exposure assessment model was developed with probability distributions used to characterise uncertainty and variability in the input data. Overall results show the mean adult human exposure to AR E. coli from tap water consumption ranged between 3.44×10-7 and 2.95×10-1cfu/day for the scenarios tested and varied depending on the water treatments used. The level of AR E. coli required in the source water pre-treatment to exceed the DWD varied between 1 and 5logcfu/ml, depending on the water treatments used. This can be used to set possible monitoring criteria in pre-treated water for potential ARB exposure in drinking water.

Fungal treatment for the removal of antibiotics and antibiotic resistance genes in veterinary hospital wastewater.

The emergence and spread of antibiotic resistance represents one of the most important public health concerns and has been linked to the widespread use of antibiotics in veterinary and human medicine. The overall elimination of antibiotics in conventional wastewater treatment plants is quite low; therefore, residual amounts of these compounds are continuously discharged to receiving surface waters, which may promote the emergence of antibiotic resistance. In this study, the ability of a fungal treatment as an alternative wastewater treatment for the elimination of forty-seven antibiotics belonging to seven different groups (ß-lactams, fluoroquinolones, macrolides, metronidazoles, sulfonamides, tetracyclines, and trimethoprim) was evaluated. 77% of antibiotics were removed after the fungal treatment, which is higher than removal obtained in conventional treatment plants. Moreover, the effect of fungal treatment on the removal of some antibiotic resistance genes (ARGs) was evaluated. The fungal treatment was also efficient in removing ARGs, such as ermB (resistance to macrolides), tetW (resistance to tetracyclines), blaTEM (resistance to ß-lactams), sulI (resistance to sulfonamides) and qnrS (reduced susceptibility to fluoroquinolones). However, it was not possible to establish a clear link between concentrations of antibiotics and corresponding ARGs in wastewater, which leads to the conclusion that there are other factors that should be taken into consideration besides the antibiotic concentrations that reach aquatic ecosystems in order to explain the emergence and spread of antibiotic resistance.

Bacteriophages as a reservoir of extended-spectrum ß-lactamase and fluoroquinolone resistance genes in the environment.

Six antibiotic resistance genes (blaCTX-M , blaSHV , blaTEM , qnrA, qnrB and qnrS) were quantified by qPCR in both phage and bacterial DNA fractions of environmental water samples in order to determine the contribution of phages to the dissemination of antibiotic resistance genes (ARGs) in the environment. Although the highest copy numbers (p <0.05) of ARGs were detected in the bacterial DNA fraction, qnrS and blaSHV genes were found in the phage DNA from all samples analysed, reaching up to 4 log10 copy numbers/mL in hospital samples. These results indicate that bacteriophages are a potential reservoir of resistance genes and may act as efficient vehicles for horizontal gene transfer.

Nitritation versus full nitrification of ammonium-rich wastewater: comparison in terms of nitrous and nitric oxides emissions.

The processes of nitritation and full nitrification of synthetic reject wastewater were compared in terms of N2O and NO emissions. Two lab-scale sequencing batch reactors (SBR1 and SBR2) were enriched with Nitrosomonas (ammonia-oxidizing bacteria) and Nitrobacter (nitrite-oxidizing bacteria), as shown by fluorescence in situ hybridization (FISH) and high-resolution 16S rRNA tag pyrosequencing. Stable conversion of ammonium to nitrite and nitrite to nitrate was achieved in SBR1 and SBR2 respectively. Biomass from SBR2 was added in SBR1 in order to achieve full nitrification. Under nitritation, 1.22% of the converted-N was emitted as N2O, and 0.066% as NO. During the transition from nitritation to full nitrification, effluent nitrite concentrations decreased but nitrogen oxides were emitted at levels similar to the nitritation period. Gas emissions decreased sharply under full nitrification conditions (0.54% N2O-N/converted-N; 0.021% NO-N/converted-N), probably as a result of the combined effect of lower nitrite and ammonium concentrations in the bioreactor.

Multidrug resistance-encoding plasmid from Aeromonas sp. strain P2G1.

A plasmid (pP2G1), which confers multidrug resistance in an environmental Aeromonas species, was completely sequenced using a shotgun approach. Plasmid pP2G1 encoded resistance to aminoglycosides and quinolones [aac(6')-Ib-cr], ß-lactams (bla(OXA-1)), chloramphenicol (catB3), macrolides [mphA-mrx-mphR], quaternary ammonium compounds (qacEΔ1), quinolones (qnrS2), rifampicin (arr-3) and sulphonamides (sul1). These findings suggest that Aeromonas species may potentially act as reservoirs of antibiotic resistance genes.

Identification and characterization of lactic acid bacteria isolated from rainbow trout, Oncorhynchus mykiss (Walbaum), with inhibitory activity against Lactococcus garvieae.

A study was conducted to evaluate the probiotic properties of endogenous rainbow trout microbiota against pathogenic Lactococcus garvieae. A total of 335 bacterial strains were isolated from rainbow trout and screened for antagonistic activity against L. garvieae using an agar spot assay. Antagonistic strains were grouped by PCR amplification of repetitive bacterial DNA elements (rep-PCR) and identified by 16S rRNA gene sequence analysis. The results revealed that the antagonistic strains belonged to the genera Lactobacillus, Lactococcus and Leuconostoc. Further probiotic characteristics, such as specific growth rate, doubling time, resistance to biological barriers, antibiotic resistance, hydrophobicity and production of antimicrobial substances, were also studied. These strains were able to survive low pH and high bile concentrations, showed good adherence characteristics and a broad spectrum of antibiotic resistance. The antagonistic efficacy was maintained after sterile filtration and was sensitive to proteinase K, indicating that proteinaceous extracellular inhibitory compounds were at least partially responsible for pathogen antagonism. Based on these results, these strains should be further studied to explore their probiotic effects in challenge experiments in vivo. This study shows clear evidence that the indigenous trout-associated microbiota may provide a defensive barrier against L. garvieae.

The effect of Pediococcus acidilactici on the gut microbiota and immune status of on-growing red tilapia (Oreochromis niloticus).

AIM: To assess Pediococcus acidilactici as a dietary supplement for on-growing red tilapia (Oreochromis niloticus). METHODS AND RESULTS: Tilapia were fed either a control diet or control diet supplemented with Ped. acidilactici at 10(7) CFU g(-1) for 32 days. Ped. acidilactici colonized the intestinal tract and significantly affected the intestinal microbial communities. PCR-DGGE revealed direct antagonism of gastric Ped. acidilactici with an endogenous uncultured bacterium during a period of reverting to nonsupplemented feeding. Light microscopy revealed that gut integrity and leucocyte levels were unaffected by Ped. acidilactici; however, blood leucocyte levels and serum lysozyme activity were elevated after 14-days' feeding. No significant improvements in growth performance were observed at the end of the trial (day 32), but survival was significantly higher in the probiotic group. CONCLUSIONS: The study demonstrates that oral supplementation of Ped. acidilactici modulates intestinal bacterial communities in on-growing red tilapia and also stimulates some aspects of the nonspecific immune response. SIGNIFICANCE AND IMPACT OF THE STUDY: To our knowledge this is the first study assessing the effects of probiotics on the gut microbiota of tilapia using culture-independent methods. Such methods are crucial to understand the mechanisms which underpin and mediate host benefits.

Host-microbiota interactions within the fish intestinal ecosystem.

Teleost fish are in direct contact with the aquatic environment, and are therefore in continual contact with a complex and dynamic microbiota, some of which may have implications for health. Mucosal surfaces represent the main sites in which environmental antigens and intestinal microbiota interact with the host. Thus, the gut-associated lymphoid tissues (GALT) must develop mechanisms to discriminate between pathogenic and commensal microorganisms. Colonization of intestinal mucosal surfaces with a normal microbiota has a positive effect on immune regulatory functions of the gut, and disturbance in these immune regulatory functions by an imbalanced microbiota may contribute to the development of diseases. Significant attention has therefore been recently focused on the role of probiotics in the induction or restoration of a disturbed microbiota to its normal beneficial composition. Given this, this article explores the fascinating relationship between the fish immune system and the bacteria that are present in its intestinal microbiota, focusing on the bacterial effect on the development of certain immune responses.

Effect of fish farming on the water quality of rivers in northeast Spain.

The effects of fish farming activities on the aquatic environment were evaluated by studying the water quality of twelve rivers located in northeast Spain. Two sampling sites were used for each river: the first sampling point was located just upstream from the fish farming facilities and the second one was downstream from fish farm effluent discharge point. In order to avoid any misinterpretation due to watershed location and seasonality, a stratified statistical analysis was performed. The results show significant decreases in pH and dissolved oxygen, in contrast to chemical oxygen demand, ammonia, phosphates and microbiological parameters, which significantly increased downstream from the fish farm discharges. Other significant variations were also found for conductivity and temperature. According to the European and local regulations concerning to support fish populations, our results fell within the allowable limits for salmonid waters. Nevertheless, we suggest that further investigations should be carried out to study the ecological interactions between farmed and wild fish populations.